The long term goal is to elucidate the interaction of the two flavoenzymes required in alpha,beta-dehydrogenation of fatty acyl CoA's in beta-oxidation with each other, with the substrate and products of the dehydrogenation and with the rest of the beta-oxidation enzymes, and to determine the mechanisms of the electron transfers involved and fate of the product. This is being approached by rapid reaction and anaerobic titration techniques (utilizing the characteristics absorbance and fluorescences of those enzymes), displacement studies, and studies of catalytic activity in dye-coupled systems. The aims of the current research are to determine where 2 epsilon to 1 epsilon transduction occurs, to probe the relationship between these flavoproteins and the rest of the beta-oxidation enzymes, to determine the redox properties of the two flavoproteins both separately and together, to probe the nature of the apparent oxidized acyl CoA complex of the short chain acyl dehydrogenase and to identify additional flavoproteins which interact with acyl CoA derivatives. In particular, absorbance changes in these enzymes during catalysis have suggested the participation of a reduced ETF-enoyl CoA complex. The formation and breakdown of this apparent complex will be studied using different chain length (and branched chain) substrates as well as different dehydrogenases. A new fluorescence assay for ETF has been developed and will be tested on different tissues. The acyl CoA dehydrogenase-substrate complex will be investigated with phenazine methosulfate to try to determine if the mechanism of electron transfer to this dye is similar to the mechanism of electron transfer to ETF. The dehydrogenase-substrate complex is also being studied by EPR techniques.